Hindawi

BioMed Research International

Volume 2021, Article ID 9884297, 12 pages
https://doi.org/10.1155/2021/9884297

Research Article
PICK1 Deficiency Exacerbates Sepsis-Associated Acute
Kidney Injury

Qian Dou ,1 Hang Tong ,2 Yichun Yang,3 Han Zhang,1 and Hua Gan 1

1
Department of Nephrology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
2
Department of Urology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
3
Department of Gastroenterology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China

Correspondence should be addressed to Hua Gan; ganhua113@sohu.com

Qian Dou and Hang Tong contributed equally to this work.

Received 27 April 2021; Revised 19 June 2021; Accepted 24 June 2021; Published 9 July 2021

Academic Editor: Dorota Formanowicz

Copyright © 2021 Qian Dou et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

We performed in vitro and in vivo experiments to explore the role of protein kinase C-binding protein 1 (PICK1), an intracellular
transporter involved in oxidative stress-related neuronal diseases, in sepsis-related acute kidney injury (AKI). Firstly, PCR, western
blotting, and immunohistochemistry were used to observe the expression of PICK1 after lipopolysaccharide- (LPS-) induced AKI.
Secondly, by inhibiting PICK1 in vivo and silencing PICK1 in vitro, we further explored the effect of PICK1 on AKI. Finally, the
relationship between PICK1 and oxidative stress and the related mechanisms were explored. We found that the expression of
PICK1 was increased in LPS-induced AKI models both in vitro and in vivo. PICK1 silencing significantly aggravated LPS-
induced apoptosis, accompanied by ROS production in renal tubular epithelial cells. FSC231, a PICK1-specific inhibitor,
aggravated LPS-induced kidney injury. Besides, NAC (N-acetylcysteine), a potent ROS scavenger, significantly inhibited the
PICK1-silencing-induced apoptosis. In conclusion, PICK1 might protect renal tubular epithelial cells from LPS-induced
apoptosis by reducing excessive ROS, making PICK1 a promising preventive target in LPS-induced AKI.

1. Introduction to understand the potential mechanism of LPS-induced renal

Acute kidney injury (AKI) is common in hospitalized patients,
with various complications and high mortality, and brings a
great challenge for clinicians [1–3]. Sepsis is the most common
cause of AKI in critically ill patients, [4] and lipopolysaccha-
ride (LPS) is an important initiating factor of sepsis, which
can induce cytokine storm, oxidative stress, hypotension,
insufficient renal perfusion, premature senescence, and even-
tually lead to the gradual decline of renal function, [5, 6] thus
participating in the occurrence and development of sepsis-
associated acute kidney injury (SA-AKI). LPS would make a
valuable contribution towards the pathogenesis of sepsis-
associated AKI. Moreover, apoptosis of renal tubular epithelial
cells (TECs) also contributes to the progress of AKI [7–9].
Apoptosis induced by endotoxin plays an important role in
sepsis-associated acute renal injury. Therefore, it is necessary
tubular cell injury and apoptosis.
Reactive oxygen species (ROS), which contained exoge-
nous oxidants and active oxygenated compounds produced
in oxidative metabolism in vivo, plays an essential role in cell
apoptosis [10, 11]. When cells are exposed to harmful stimu-
lation, a lot of ROS will be produced, leading to the oxidant-
antioxidant imbalance [12–14]. Recent studies have shown
that oxidative stress is the leading cause of LPS-induced
mitochondrial damage in renal cells [15], and the mitochon-
drial dysfunction in turn triggers apoptosis by activating the
cascade of tandem proteases [16–19]. Therefore, reducing
oxidative stress may be an effective way to decrease LPS-
induced apoptosis of TECs.
Protein interacting with C-kinase 1 (PICK1) is a unique
protein containing both BAR (Bin/Amphiphysin/Rvs) and
PDZ (PSD-95/DlgA/ZO-1) domains. This particular struc-
2 BioMed Research International
ture enables PICK1 to bind to various membrane proteins,
thus initiating multiple functions [20–22]. PICK1 is widely
expressed in various organs and has been found to participate
in the oxidative regulation by regulating glutathione (GSH,
an important antioxidant) [23] in the lung [24] and liver
[25]. However, the research on the relationship between
PICK1 and SA-AKI is relatively rare.
In our study, using a septic mouse model by injecting
LPS, we investigated the role of PICK1 in the pathophysio-
logical process of septic-induced AKI and explored the
underlying mechanism.
2. Materials and Methods
2.1. Animal Models and Groups. Our animal experiment was
carried out in the Animal Laboratory Center of Chongqing
Medical University (Chongqing, China). Male C57BL/6 mice
(6–8 weeks old, 20–25 g) purchased from the Animal Labora-
tory Center of The First Affiliated Hospital of Chongqing
Medical University were used in this study. The Animal
Experimentation Ethics Committee of The First Affiliated
Hospital of Chongqing Medical University approved all
animal experiments. Mice were housed under specific
pathogen-free conditions, and the standard rodent chow
and water were available ad libitum. The septic AKI animal
model was established by intraperitoneal injection of 10
mg/kg of LPS for 24 hours [15, 26].
FSC231 (529531, Merck, Germany), a specific PICK1
inhibitor [27], was given to examine the role of PICK1 in
LPS-induced AKI. C57BL/6 mice were randomly divided
into three groups (n = 6 in each group): the control group,
LPS group, and LPS+FSC231 group. The LPS group was
given intraperitoneal injection of LPS, and the control group
was given the same dose of saline. The LPS+FSC231 group
was injected with FSC231 (78.4 μg/g) for five consecutive
days, followed by LPS administration 2 hours after the last
dose of FSC231 [25]. Renal tissue and blood samples were
collected after 24 hours of LPS treatment.
2.2. Assessment of Kidney Function. Blood samples were col-
lected from the eyeballs. According to the manufacturer’s
illustration, blood urea nitrogen (BUN) and serum creatinine
(Scr) were measured enzymatically.
2.3. Histology Analyses. Kidney specimens were fixed with 4%
paraformaldehyde, embedded in paraffin, sectioned into 4
μm thick, and stained with hematoxylin and eosin. The path-
ological changes of renal tissue were observed under the light
microscope. A semiquantitative scoring method was used to
evaluate the degree of renal tubular injury: 0 for less than
5%, 1 for 5%-25%, 2 for 25%-50%, 3 for 50%-75%, and 4
for more than 75%. Ten areas in each section were randomly
observed under the light microscope (400x magnification) to
evaluate tubular epithelial necrosis, lumen dilatation, and
tubular injury.
2.4. Immunohistochemistry. The expression of PICK1 and
cleaved caspase 3 were detected by immunohistochemistry.
The dewaxed slices were put into citrate buffer (0.01 M, pH
6.0) and heated in microwave oven for 20 minutes. Then,
we complete the remaining dyeing steps according to the
manufacturer’s immunoassay kit instructions (ZSGB-BIO
Technology Co., Ltd., Beijing, China). PICK1 (10983-2-AP,
Proteintech, China) and cleaved caspase 3 (19677-1-AP,
Proteintech, China) were used as the primary antibody at a
dilution of 1 : 200. The images were taken with a digital
camera under a microscope.
2.5. Cell Culture and Treatment. The human renal proximal
tubular epithelial cell line (HK2) was presented by Dr. Jiang,
Center for kidney disease, Chongqing Medical University.
The cells were cultured in 1640 medium (Gibco, USA)
containing 10% FBS (PAN-Biotech, Adenbach, Germany)
and 1% penicillin-streptomycin (Beyotime, China) at 37°C
in 5% CO2 wet air with or without Lenti-NC and Lenti-
PICK1 to silence the expression of PICK1 (NC and sh-
PICK1). And then, for the treatment with LPS (15 μg/ml)
for 24 hours, the cells were divided into the WT group,
WT+LPS group, NC+LPS group, and sh-PICK1+LPS group
for further experiments.
2.6. Transfection. Lenti-NC and Lenti-PICK1 were built at
Shanghai Genechem Co., Ltd. (Shanghai, China). HK2 cells
were seeded into 6-well plates. When the cell fusion reached
50%, the medium containing lentivirus and HitransG A was
added. After 24 hours of incubation, the medium was
changed, and the cells were cultured for another 24 hours
for further analysis. Besides, stably transfected cell lines were
screened by puromycin (2 ng/ml). Then, cells were cultured
with the optimum concentration of LPS for the given time.
2.7. Immunofluorescence Staining. HK2 were inoculated in 24
aperture plates after the cells reached 40% to 50% confluency,
treated with LPS for 24 hours after adhering the wall, fixed
with 4% paraformaldehyde for 15 minutes at room tempera-
ture, and then immersed with 0.1% TritonX-100 (p0096,
Jiangsu, China) and 5% goat serum for 15 min and 30 min,
respectively. After culture with PICK1 antibody (1 : 200, Pro-
teintech, China) at 4°C overnight, goat anti-rabbit IgG (1 : 20,
Proteintech, China) was added in the dark for 1 hour. The
cells were then stained with 2-(4-aminophenyl)-6-indolea-
mine dihydrochloride (DAPI) for 10 minutes. Zeiss LSM
800 confocal laser scanning microscope (Carl Zeiss,
Germany) was used for image acquisition and observation.
2.8. GSH Measurement. According to the manufacturer’s
illustration, GSH-Px (Total Glutathione Peroxidase Test
Kit, S0053, Beyotime, China) was determined with the corre-
sponding test kit.
2.9. Detection of Intracellular ROS Levels. According to the
manufacturer’s illustration, ROS was determined with the
corresponding test kit (Reactive Oxygen Species Assay Kit,
S0053, Beyotime, China). In short, the treated cells were
incubated with DCFH-DA (10 μmol/l) in the dark at 37°C
for 30 minutes and then washed with serum-free medium.
The fluorescence was observed under flow cytometry. The
excitation and emission wavelengths were 488 nm and
525 nm, respectively.
BioMed Research International
2.10. TUNEL Staining. According to the manufacturer’s
instructions, the apoptotic cells in renal tissue were evaluated
with the TACS TdT In Situ Apoptosis Detection kit (Roche,
Germany). Renal tissue sections were deparaffinized and
hydrated as per the standard procedure and digested with
proteinase K solution at 37°C for 30 min. After the endoge-
nous peroxidase activity was quenched, the sections were
incubated with TdT labeling reaction mixture at 37°C for
one hour and then treated with streptavidin horseradish per-
oxidase. Subsequently, the prepared samples were washed
with phosphate buffer for colorimetric analysis. An optical
microscope was used to analyze the results.
2.11. Hoechst 33258 Staining. According to the manufac-
turer’s instructions, the cells were washed twice with PBS.
Then, the samples were fixed with 4% formaldehyde for
15 min at room temperature and stained with Hoechst
33258 (Beyotime, China) solution in the dark for 5 minutes.
After incubation, the apoptotic cells were observed under a
fluorescence microscope.
2.12. Real-Time Quantitative Polymerase Chain Reaction
(RT-PCR). According to the manufacturer’s instructions,
cell RNA was extracted with TRIzol reagent (Invitrogen,
Shanghai, China). Total RNA was reverse transcribed into
cDNA by Prime Script RT Kit (Takara Biotechnology Inc.,
Shiga, Japan). RT-qPCR was performed with the following
primer pairs:
PICK1-F 5 ′ -AGTTCGGCATTCGGCTTC-3 ′ , PICK1-R
5 ′ -GAAGCCGAATGCCGAACT-3 ′ , GAPDH-F 5 ′ -GGTG
AAGGTCGGAGTCAACG-3 ′ , and GAPDH-R 5 ′ -CAAA
GTTGTCATGGATGHACC-3 ′ .
The target gene level was normalized with GAPDH level,
and the mRNA level was calculated by the standard method.
2.13. Western Blot. RIPA buffer containing PMSF and phos-
phatase inhibitors was used to extract the proteins of renal
tissue and HK2 cells protein (all from Beyotime, China).
According to the manufacturer’s protocol, the protein
concentration was determined by the BCA protein assay
kit. The protein samples of each group were separated by
SDS-PAGE and transferred to PVDF membranes (MILI
Bloomberg, Massachusetts). The PVDF membranes were
sealed with rapid blocking solution at room temperature.
The primary antibodies included rabbit anti PICK1 (1 : 500,
protein tech,) rabbit anti Cleaved Caspase-3, rabbit anti
Bax, and rabbit anti BCL-2 (1 : 500, Proteintech, China), rab-
bit anti ASK1 (1 : 500,Proteintech, China), rabbit anti
p38MAPK (1 : 500, Proteintech, China), and rabbit anti
GAPDH (1 : 8000; Proteintech, China). Using the HRP-
labeled secondary antibody (1 : 3000 dilution) to incubate
the membrane at 37°C for 1 hour. Finally, the fusion imaging
system is used to detect the relative densities of the bands.
2.14. Flow Cytometry. Annexin V-FITC/PC was used to
detect apoptosis with the test kit (Beyotime, China) accord-
ing to the manufacturer’s instructions. The apoptosis was
analyzed by flow cytometry (BD Biosciences, USA).
3
2.15. CCK-8. According to the manufacturer’s illustration,
the proliferation of pretreatment cell samples was detected
by the CCK-8 kit (C0038, Beyotime, China). We add 10 μl
reagent to each sample for two hours and then detect the
absorbance at 450 nm wavelength.
2.16. Statistical Analysis. All data were expressed as the
means ± standard deviation and analyzed by GraphPad
Prism 9.0 software using one-way ANOVA or t-test. Differ-
ences were considered significant at P < 0:05.
3. Results
3.1. PICK1 Was Increased in the LPS-Induced Renal Proximal
Tubular Cells. Western blotting and qPCR were used to inves-
tigate the effect of LPS on the expression of PICK1 in vitro. We
found that the expression of PICK1 increased after LPS treat-
ment at different concentrations (0, 5, 15, and 20 μg/ml,
Figures 1(a) and 1(b)). In HK2 cells, PICK1 gradually increased
with time after LPS treatment (15 μg/ml, Figure 1(c)). During
our observation period, the expression of PICK1 reached the
peak after treatment with 15 μg/ml LPS for 24 hours. Therefore,
LPS induced the increasing expression of PICK1 protein in a
time- and dose-dependent manner.
The level of PICK1 in LPS-treated mouse kidney was also
detected in vivo. After treatment with LPS (10 mg/kg) for 24
h, the expression of PICK1 in the renal cortex and medulla
significantly increased as showed in the immunohistochemi-
cal staining image (Figures 1(d) and 1(e)). The bands of
western blotting also showed that PICK1 in the kidney tissue
of LPS-induced AKI mice was increased, and FSC231
decreased the level of PICK1 (Figure 1(f)).
3.2. Inhibition of PICK1 Aggravated the LPS-Induced AKI.
FSC231, acting as a particular PICK1 inhibitor [27], was
administered to investigate the role of PICK1 in LPS-
induced AKI. The immunohistochemistry results showed
that FSC231 reduced the PICK1 expression in LPS-induced
AKI (Figures 2(a) and 2(b)). Both BUN and serum creatinine
(Scr) presented a rapid increase after FSC231 treatment
(Figures 2(c) and 2(d)). Histological analysis revealed that
the expression of PICK1 in the kidney tissue of LPS-
induced AKI mice was increased, and FSC231 treatment
aggravated AKI, which was reflected by the exfoliated TECs,
the damaged tubular structure, and the necrotic epithelial
cells (Figures 2(e) and 2(h)). The number of TUNEL-
positive cells in the kidney of mice pretreated with PICK1
inhibitor FSC231 was also increased (Figures 2(f) and 2(g)).
Therefore, the inhibition of PICK1 aggravated the LPS-
induced AKI in vivo. However, the appeal results all show
that using FSC231 alone without LPS have no significant
effect on tissue morphology and function.
3.3. PICK1 Silencing Promoted TEC Apoptosis. Compared
with the control group, after 24 hours of transfecting, PICK1
shRNA significantly reduced both the mRNA and protein of
PICK1 (Figures 3(a) and 3(b)). The proapoptotic protein Bax
and cleaved-caspase 3 was upregulated while the antiapopto-
tic protein Bcl-2 was downregulated after PICK1 silencing
(Figure 3(c)). Similarly, the expression of cleaved-caspase 3
4 BioMed Research International

LPS (μg/ml) 0 5 15 20
PICK1 50kDa
2.5 ns Time (h) 0 12 24 48
⁎⁎⁎ GAPDH 36kDa PICK1 50kDa
2.0 2.0 GAPDH 36kDa
Relative PICK1 level

ns 2.0 ns
⁎

Protein related level

(Protein/GAPDH)
⁎⁎⁎

Protein-related level
1.5 ⁎⁎⁎

(Protein/GAPDH)
1.5
1.5
1.0 1.0
1.0
ns ⁎
0.5 0.5 0.5

0.0 0.0 0.0

LPS (μg/ml) 0 5 15 20 PICK1 PICK1

LPS (μg/ml) 0h 24h

0 15 12h 48h
5 20
(a) (b) (c)
LPS - + +
FSC231 - - +

60 PICK1-Vivo PICK1
⁎⁎ Vivo
GAPDH
⁎⁎⁎
PICK1 IHC score

40 1.0 ⁎⁎

Protein-related level
(Protein/GAPDH)
0.8
PICK1-Vivo
0.6
20
0.4
0.2
20um 20um
0 0.0
Control LPS Control LPS PICK1
Control
LPS
LPS+FSC231
(d) (e) (f)

Figure 1: PICK1 was increased in LPS-induced AKI models both in vitro and in vivo. (a, b) PCR and western blotting results after treatment
with different concentrations of LPS for 24 hours (0 μg/ml versus 5 μg/ml, 0 μg/ml versus 15 μg/ml; ∗ P < 0:05, ∗∗∗ P < 0:001). (c) Western
blotting results after LPS treatment for different times (0 h versus 12 h, 0 h versus 24 h; ∗ P < 0:05, ∗∗∗ P < 0:001). (d, e) Representative
immunohistochemical images of pretreated renal tissue (magnification ×400, scale = 20 μm, the control versus LPS, ∗∗ P < 0:01). (f)
Representative western blotting analysis showed the expression of PICK1 in the kidney of mice treated with LPS (10 mg/kg) and FSC231
(78.4 μg/g) (the control versus LPS, ∗∗ P < 0:01).

in the LPS+FSC231 group was significantly increased in trations (Figure 3(i)). These results indicated that PICK1 was
immunohistochemistry, but in the case of using FSC231 involved in the proapoptotic effect of LPS on HK2 cells.
alone, the expression of cleaved-caspase 3 did not change sig-
nificantly (Figure 3(d)), suggesting that under the conditions 3.4. PICK1 Inhibition Increased the Production of Peroxide
of LPS-induced septic kidney injury, PICK1 inhibition aggra- and Activated the ASK1-p38 Apoptotic Pathway after LPS
vated the apoptosis of renal cells. The immunofluorescence Treatment. As an important intracellular regulatory metabo-
results of HK2 cells showed that LPS could increase the lite, GSH could act as a direct antioxidant to participate in
expression of PICK1, but shRNA could decrease PICK1 biotransformation. We detected the changes in GSH and
(Figures 3(e) and 3(g)). Hoechst 33258 staining showed that ROS content after LPS pretreatment. GSH measurement
PICK1 silencing evidently exacerbated LPS-induced apopto- result showed that the content of GSH in the PICK1 inhibi-
sis (Figures 3(f) and 3(h)). Moreover, the CCK-8 test showed tion group was significantly lower than that in the NC group
that the proliferation of cells in the PICK1 knockdown group both in vivo and in vitro (Figures 4(a) and 4(b)). The results
was significantly slowed down at different times and concen- of flow cytometry indicated that the peroxide product in the
BioMed Research International 5

60 PICK1-Vivo

PICK1 IHC scores

PICK1-Vivo
40 ⁎

20

20um 20um
0
LPS LPS+FSC231
LPS LPS+FSC231
(a) (b)
60 BUN 30 Scr
⁎⁎ ⁎⁎

⁎ ⁎⁎
40 20
BUN (mmol/l)

Scr (μmol/l)
ns ns

20 10

0 0
Control FSC231 LPS LPS+231 Control FSC231 LPS LPS+231

(c) (d)
LPS - - + +
FSC231 - + - +

HE

20um 20um 20um 20um

(e)
LPS - - + +
FSC231 - + - +

TUNEL

20um 20um 20um 20um

(f)
250 5
⁎⁎ ⁎⁎⁎
TUNEL-positive nuclei

200 4
Tubular injury scores

⁎⁎⁎ ⁎⁎⁎
150 3

100 2
ns ns
50 1

0 0
Control FSC231 LPS LPS+Fsc231 Control FSC231 LPS LPS+FSC231

(g) (h)

Figure 2: PICK1 inhibition aggravated LPS-induced AKI. (a, b) The results of immunohistochemistry (magnification ×400, scale = 20 μm,
LPS versus LPS+FSC231, ∗ P < 0:05). (c) The results of BUN (the control versus LPS, LPS versus LPS+FSC231; ∗∗∗ P < 0:001). (d) The
results of Scr (the control versus LPS, LPS versus LPS+FSC231, ∗∗ P < 0:01). (e) Hematoxylin-eosin (H & E) staining showed the renal
histological damage (magnification ×400, scale = 20 μm). (f, g) Representative images and statistical analysis of TUNEL-positive cells
(magnification ×400, scale = 20 μm, the control versus LPS, LPS versus LPS+FSC231; ∗∗ P < 0:01, ∗∗∗ P < 0:001). (h) The results of tubular
injury scores (the control versus LPS, LPS versus LPS+FSC231, ∗∗∗ P < 0:001).
6 BioMed Research International

1.5

Relative PICK1 level

1.0 1.5
⁎⁎

Protein-related level
(Protein/GAPDH)
1.0
0.5
WT NC sh-PICK1 0.5
PICK1 50kDa

GAPDH 36kDa 0.0

0.0
PICK1
WT NC sh-PICK1
WT
NC
sh-PICK1

(a) (b)
LPS - - + +
FSC231 - + - +
WT NC sh-PICK1
Bax 21kDa

Cleaved- 19kDa
caspase 3 17kDa 20um 20um 20um 20um

Bcl-2 26kDa Cleaved-caspase 3

80 ⁎⁎
GAPDH 36kDa
Cleaved-caspase 3 IHC scores

2.5
⁎⁎⁎ 60
⁎⁎⁎
Protein-related level

2.0
(Protein/GAPDH)

⁎⁎⁎ ⁎⁎⁎
1.5 40
ns
ns
1.0
20
ns ns
0.5

0.0 0
Bax Cleaved- Bcl-2 Control FSC231 LPS LPS+FSC231
caspase 3

WT
NC
sh-PICK1

(c) (d)

WT WT+LPS NC+LPS sh-PICK1+LPS

(e)

Figure 3: Continued.
BioMed Research International 7

10 ⁎⁎⁎
ns

PICK1 relative red fluorescence

8

0
WT WT+LPS NC+LPS sh-PICK1+LPS

PS
LP

LP
W

+L
T+

C+

K1
W

IC
-P
Sh
(f) (g)
80
⁎⁎⁎

1.0
60
Cell viability (OD 450nm)
Apoptosis rate (%)

Cell viability (% of control)

0.8 120
40 100
0.6
ns 80
0.4 60
20
40
0.2
20
0 0.0 0
12 24 48 72 0 5 10 15
T

PS
LP

LP
W

+L
T+

C+

K1

Time (hours) LPS Concentration (μg/ml)

W

IC
-P
Sh

Control Control
NC NC
sh-PICK1 sh-PICK1
(h) (i)

Figure 3: Downregulation of PICK1 aggravated LPS-induced apoptosis of HK2 cells. (a, b) The levels of PICK1 mRNA and protein were
detected by qPCR and WB (NC versus sh-PICK1, ∗∗ P < 0:01). (c) The expression levels of apoptosis-related proteins Bcl-2, Bax, and
cleaved caspase 3 were detected by western blotting (NC versus sh-PICK1, ∗∗∗ P < 0:001). (d) Representative cleaved caspase 3 level
detected by immunohistochemistry (the control versus LPS, LPS versus LPS+FSC231; ∗∗ P < 0:01, ∗∗∗ P < 0:001). (e, g) Representative
immunofluorescence results (WT+LPS versus NC+LPS, NC+LPS versus sh-PICK1+LPS; ∗∗∗ P < 0:001). (f–h) The apoptosis of HK2 cells
was detected by Hoechst 33258 staining (magnification ×200, scale = 50 μm, WT+LPS versus NC+LPS, NC+LPS versus sh-PICK1
+LPS; ∗∗∗ P < 0:001). (f, h) Representative Hoechst 33258 staining results (WT+LPS versus NC+LPS, NC+LPS versus sh-PICK1+LPS;
∗∗∗
P < 0:001). (i) The apoptosis of HK2 cells was detected by CCK-8.

sh-PICK1 group was significantly higher than that in the NC (Figures 5(a) and 5(b)). Additional pretreatment with 10
group (Figure 4(c)). Previous studies have shown that ROS mM NAC for one hour significantly downregulated the
can initiate apoptosis by activating the ASK1-p38MAPK PICK1-silencing-exacerbated ROS and TEC apoptosis
signaling pathway [44]. Our western blotting showed that (Figure 5(c)–5(e)). At the same time, the expression of
PICK1 inhibition upregulated the expression of ASK1 and ASK1 and p38MAPK was decreased, and the proapoptotic
p38MAPK both in the HK2 and mouse model, suggesting protein Bax and cleaved-caspase 3 was downregulated while
that PICK1 inhibition could activate the ASK1-p38MAPK the antiapoptotic protein Bcl-2 was upregulated after NAC
signaling pathway (Figures 4(d) and 4(e)). Therefore, PICK1 pretreatment detected by western blotting (Figures 5(f) and
silencing might be involved in LPS-induced ROS production 5(g)). Based on our results, ROS-mediated apoptosis may
and the activation of the ASK1-p38MAPK signaling path- participate in PICK1-inhibition-induced HK2 cell apoptosis
way in vitro. through activating the ASK1-p38MAPK signaling pathway.

3.5. NAC Inhibited the LPS-Induced Apoptosis and ROS. 4. Discussion

NAC is an important scavenger for oxidation products
in vivo [15]. We further explored the role of NAC on LPS- Acute kidney injury (AKI) is a significant challenge for clini-
induced apoptosis in TECs. Flow cytometry showed that cians, and sepsis-associated acute kidney injury (SA-AKI)
PICK1 silencing exacerbated LPS-induced early apoptosis has been proved to increase the mortality of children and
8 BioMed Research International

150 Vitro
60 Vivo

GSH concentration
ns

GSH concentration
100 ⁎⁎⁎
40 ⁎

(μm)

(μM)
50 20
⁎⁎⁎

0 0
NC sh-PICK1

31

31
ro

LP
C2

C2
nt
Co

FS

FS
l+

S+
ro

LP
nt
Co
(a) (b)
Vitro
WT NC sh-PICK1
ASK1 155kDa

p38MAPK 38kDa

GAPDH 36kDa
2.0 ⁎⁎⁎
400 400
Protein-related level ⁎⁎⁎
(Protein/GAPDH)
1.5
Count

Count

200 200 1.0

0.5 ns ns

0 0 0.0
102 103 104 105 106 107 102 103 104 105 106 107 ASK1 p38MAPK
DCFH FITC-A DCFH FITC-A
WT
NC sh-PICK1 NC
sh-PICK1
(c) (d)
2.0
⁎⁎⁎
Protein-related level
(Protein/GAPDH)

Vivo 1.5 ⁎⁎
LPS - - + +
FSC231 - + - + ⁎⁎⁎ ⁎⁎⁎
1.0
ASK1 155kDa
0.5
ns ns
p38MAPK 38kDa
0.0
ASK1 p38MAPK
GAPDH 36kDa
Control LPS
FSC231 LPS+FSC231
(e)

Figure 4: Knockdown of PICK1 increased the production of peroxide and activated the apoptotic pathway after LPS treatment. (a) After
treatment with LPS for 24 hours, the content of GSH in the NC and sh-PICK1 groups (NC versus sh-PICK1, ∗∗∗ P < 0:001). (b) The
content of GSH in each group of mouse model (LPS versus LPS+FSC231, ∗∗∗ P < 0:001). (c) Representative fluorescence images of
ROS. (d, e) Representative western images of the ASK1 and p38MAPK pathway (NC versus sh-PICK1, ∗∗ P < 0:01; ASK1: the
control versus LPS, ∗∗∗ P < 0:001; LPS versus LPS+FSC231, ∗∗ P < 0:01; p38MAPK: the control versus LPS, ∗∗∗ P < 0:001; LPS versus
LPS+FSC231, ∗∗∗ P < 0:001).

adults [1–3]. Endotoxin, especially lipopolysaccharide (LPS), nism of LPS-induced AKI, there is no effective intervention
is a common cause of SA-AKI [5]. However, since the lack of so far. PICK1 has been reported to have an antioxidant effect
understanding of the complex pathophysiological mecha- in the nervous system [23], but the role of PICK1 in AKI has
BioMed Research International 9

Q1-UL (1.21%) Q1-UR (6.60%) Q1-UL (0.73%) Q1-UR (9.43%) Q1-UL (0.61%) Q1-UR (12.91%) Q1-UL (2.84%) Q1-UR (23.10%)
106 106 106 106
DAPI PB450-A

DAPI PB450-A

DAPI PB450-A

DAPI PB450-A
105 105 105 105

104 104 104 104

103 103 103 103

Q1-LL (89.08%) Q1-LR (3.11%) Q1-LL (78.82%) Q1-LR (11.02%) Q1-LL (75.61%) Q1-LR (10.87%) Q1-LL (67.22%) Q1-LR (6.84%)

103 104 105 106 103 104 105 106 103 104 105 106 103 104 105 106
Annexin V APC-A Annexin V APC-A Annexin V APC-A Annexin V APC-A
WT WT+LPS NC+LPS sh-PICK1+LPS
(a)

0.4 400
Apoptosis 300
⁎⁎
Apoptotic rate (%)

0.3
200
⁎⁎
Count

Count
0.2 200

100
0.1

0.0 0 0
102 103 104 105 106 107 102 103 104 105 106 107
T

PS
LP

LP
W

+L

DCFH FITC-A DCFH FITC-A

T+

C+

K1
W

IC

sh-PICK1+LPS sh-PICK1+LPS+NAC
-P
sh

(b) (c)
0.3

Q1-UL (2.58%) Q1-UR (20.12%) Q1-UL (1.23%) Q1-UR (11.67%)

106 106
Apoptotic rate (%)

0.2
DAPI PB450-A

DAPI PB450-A

105 105 ⁎⁎⁎

104 104
0.1

103 103
Q1-LL (70.45%) Q1-LR (6.85%) Q1-LL (84.21%) Q1-LR (2.89%)
0.0
103 104 105 106 103 104 105 106
PS

AC

Annexin V APC-A Annexin V APC-A

+L

+N
K1

PS
IC

sh-PICK1+LPS sh-PICK1+LPS+NAC
+L
-P

K1
sh

IC
-P
sh

(d) (e)
1.0
Protein-related level
(Protein/GAPDH)

Vitro 0.8
sh-PICK1 + +
LPS + + 0.6 ⁎⁎
NAC - + 0.4 ⁎⁎⁎
ASk1 155kDa 0.2

p38MAPK 38kDa 0.0

ASK1 p38MAPK
GAPDH 36kDa
sh-PICK1+LPS
sh-PICK1+LPS+NAC
(f)

Figure 5: Continued.
10
Vitro
Protein-related level
(Protein/GAPDH)
sh-PICK1 + +
LPS + +
NAC - +
Bax 21kDa
Cleaved- 19kDa
caspase 3
17kDa
Bcl-2 26kDa
GAPDH 36kDa
(g)
Figure 5: Inhibition of PICK1 expression could increase LPS-induced apoptosis, and NAC could alleviate pretreatment-induced apoptosis.
(a, b) After 24 hours of LPS treatment in the different groups, apoptosis rate was detected by flow cytometry (WT versus WT+LPS, NC+LPS
versus sh-PICK1+LPS; ∗∗ P < 0:01). (c–e) HK2 cells were pretreated with 10 mM NAC for 1 h and then treated with LPS for another 24 h.
Representative images of ROS and apoptosis rate in flow cytometry after NAC and LPS treatment (sh-PICK1+LPS versus sh-PICK1+LPS
+NAC, ∗∗∗ P < 0:001). (f) After treatment with NAC, the ASK1 and p38MAPK were detected by western blotting (sh-PICK1+LPS versus
sh-PICK1+LPS+NAC; ∗∗ P < 0:01, ∗∗∗ P < 0:001). (f) After treatment with NAC, the apoptotic protein was detected by western blotting
(sh-PICK1+LPS versus sh-PICK1+LPS+NAC, ∗∗∗ P < 0:001).
barely been reported. Thus, we investigated whether PICK1
played a vital role in LPS-induced AKI in vivo and in vitro
and found that PICK1 deficiency exacerbated SA-AKI.
Apoptosis is a programmed cell death necessary for cellu-
lar homeostasis [28]; however, excessive apoptosis can also
cause tissue damage [29, 30]. The regulation of apoptosis
involves many steps, and there are 3 main apoptosis regulat-
ing pathways as follows: the exogenous pathway, in which the
death receptor pathway is a very important part [31]; in the
intrinsic pathway, in which the mitochondrial apoptotic
signaling pathway plays a pivotal role [32]; and in the endo-
plasmic reticulum stress-induced pathway, in which ROS
destroyed the ER function and initiated unfolded protein
response (UPR) and endoplasmic reticulum stress (ERS)
in vivo and vitro, which might be an important mechanism
that lead to tissue damage and cell apoptosis [33]. This study
found that PICK1 inhibitor FSC231 significantly promoted
the apoptosis of renal tubular epithelial cells in vivo and
aggravated the LPS-induced renal injury. However, the use
of FSC231 alone has no obvious effect on the functions of
the kidney tissue. Therefore, PICK1 has a protective effect
on AKI induced by LPS.
Oxidative stress is an inevitable reaction in life [34].
Various harmful stimuli can break the balance of oxidative
stress, leading to cell apoptosis and pathological damage
[12–14]. Previous studies have shown that oxidative stress
played a vital role in the pathogenesis of septic kidney injury
[15]. Oxidative stress and its induced apoptosis were con-
firmed to be essential pathogenic factors in chronic renal fail-
ure (CRF), [35] renal ischemia-reperfusion injury(IR) [36],
acute renal injury (AKI) [37], and diabetic nephropathy
(DN) [38]. Among various mechanisms, the endogenous
pathway of mitochondrial initiation; the regulation of the
Bcl-2 family, NF-κB, and MAPK family; and the activation
of caspase were most closely related to oxidative stress-
induced apoptosis [39]. In our septic AKI model, we found
that oxidative stress was the main factor leading to mito-
BioMed Research International
1.5
⁎⁎⁎
1.0
⁎⁎⁎
0.5
⁎⁎⁎
0.0
Bax Cleaved- Bcl-2
caspase 3
sh-PICK1+LPS
sh-PICK1+LPS+NAC
chondrial dysfunction, which caused a large-scale production
of reactive oxygen species (ROS). JNK and p38MAPK,
members of the MAPKs family, could transmit apoptosis
signal to the mitochondria, release cytochrome C, activate
specific caspase enzyme, and induce apoptosis.
Previous studies have shown that PICK1 interacts with
various neurotransmitter receptors, enzymes, and trans-
porters through its unique structure to affect synaptic
function, leading to nerve damage, such as epilepsy and Par-
kinson’s disease [22, 23, 40]. PICK1 was also involved in
breast cancer by inhibiting TGF-β signaling, thus initiating
early cancer [41]. Besides, PICK1 deficiency aggravated
sepsis-induced acute lung injury through lysosomal injury
[24, 42]. And PICK1 may mediate the prosurvival activity
of PKCalpha by serving as a molecular link between PKCal-
pha and the mitochondria, which results in a more stable
mitochondrial membrane potential, enhances phosphoryla-
tion of the anti-apoptotic Bcl-2 protein, and decreases
dimerization of the.
proapoptotic Bax protein [45]. In other words, PICK1
was involved in the pathophysiological changes of many
diseases. Recently, PICK1 has been reported to regulate glu-
tathione (GSH) homeostasis, indicating that PICK1 may play
a key role in oxidative stress [23]. However, the relationship
between PICK1 and LPS-induced AKI remains unclear.
In our research, PICK1 acted as a negative regulator of
ROS production in the mitochondria by regulating GSH
content and influenced the intrinsic apoptotic pathway to
improve cell survival. We used sh-PICK1 to inhibit PICK1
expression in HK2 to further explore whether PICK1 could
protect TECs in septic conditions. Our results demonstrated
that PICK1 was upregulated after LPS treatment. Silencing
PICK1 increased cell and tissue damage. According to the
Hoechst staining, flow cytometry, and western blotting
results, knockout of PICK1 raised LPS-induced ROS produc-
tion and aggravated the apoptosis of renal TECs. We further
confirmed that ROS production activated the ASK1-
BioMed Research International
p38MAPK apoptosis pathway and eventually led to cell and
tissue damage. Therefore, PICK1 may play an essential role
in LPS-induced renal TEC apoptosis. Similarly, immunohis-
tochemistry and pathological sections showed that FSC231
could increase the expression of an apoptotic protein in renal
cells and aggravate renal tissue injury. Both in vitro and
in vivo experiments showed that silencing PICK1 reduced
the production of GSH but increased the ROS in the LPS-
induced AKI model. NAC is a kind of ROS scavenger [15,
43]. Pretreatment with NAC significantly decreased apopto-
sis and ROS and alleviated the PICK1-inhibition-induced
injury of HK2 cells. Based on these results, PICK1 silencing
upregulated ROS production by decreasing GSH content,
[26] which might explain the mechanism of PICK1-
mediated cell survival.
In conclusion, we first demonstrated that the highly
expressed PICK1 after LPS-induced AKI might be an
endogenous protective factor. Simultaneously, the ASK1-
p38MAPK pathway might be involved in the promotion
of PICK1-deficiency-induced apoptosis. Specifically, the
silencing of PICK1 increased the production of ROS and
activated the apoptotic pathway, thus aggravating the apo-
ptosis of HK2 cells. Therefore, PICK1 may be a promising
preventive target in LPS-induced AKI.
Data Availability
All supporting data are included within the main article.
Conflicts of Interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that
could be construed as a potential conflict of interest.
Authors’ Contributions
GH and DQ designed the research and revised the manuscript.
DQ and TH performed the experiments and wrote the draft
manuscript. DQ, ZH, YYC and TH analyzed the experimental
results. All authors read and approved the manuscript and
agree to be accountable for all aspects of the research in ensur-
ing that the accuracy or integrity of any part of the work are
appropriately investigated and resolved. Qian Dou and Hang
Tong are co-first authors of the article.
Acknowledgments
We appreciate the Nephrology Department of The First
Affiliated Hospital of Chongqing Medical University for the
technical support.
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